In the preparation of recombinant insulins, the microbial biosynthesis of the insulin takes place in bacteria such as transformed Escherichia coli via a single-chain precursor molecule which, besides the natural insulin sequence of the A and B chains, comprises a connecting peptide and a fusion protein sequence. The latter is connected to the N-terminal end of the complete protein for genetic engineering reasons, and is responsible for the expressed insulin-containing product resulting in the form of inclusion bodies in the transformed E. coli. The aim of the working up is to make the insulin available from this fusion protein in multistage protein-chemical process steps. This entails in every case, just like the natural biosynthesis of insulin in the beta cell of the pancreas, passing through the stage of folding to (pre)proinsulin. The (pre)proinsulin is converted by enzymatic cleavage (for example, with trypsin) into a cleavage mixture which comprises another insulin precursor, which is di-Arg-(B31-32)-insulin, mono-Arg-B31-insulin and the connecting peptide (up to 35 amino acid residues). Also produced are byproducts which are generated by the protease activity of the trypsin, such as incomplete intermediates, de-Thr-B30-insulin or else Arg-A0-insulin and preinsulin. U.S. Pat. No. 5,101,013 discloses the separation of said mixtures of insulin and insulin derivatives by atmospheric pressure or medium pressure chromatography on strongly acidic ion exchangers such as S-SEPHAROSE.RTM., FRAKTOGEL.RTM.TSK or SP TRISACRYL.RTM.. The known chromatography materials are not pressure-stable and become compressed in the chromatography columns under a pressure of above 1 MPa. When the materials become compressed, [and] separation of the insulin-containing mixtures is then no longer possible.
Another known process for isolating insulin is high-pressure liquid chromatography on lipophilically modified silica gel (U.S. Pat. No. 5,245,008 and European Patent No. 0 547 544).
Cationic exchange purification processes using atmospheric pressure do not achieve, even with optimized traditional gel materials, the separation efficiencies necessary to attain the required degree of purification for the recombinant insulin. Chromatography with modern preparative gel materials, for example POROS 50 .mu.m/Perseptiv, Source 30 .mu.m/Pharmacia or MAKROPEP 50 .mu.m/BioRad, is carried out in a medium pressure process. Medium pressure chromatography with gels of smaller particle size (for example, Source 15 .mu.m) affords only slight selectivity improvements in respect of the separation efficiency, so that, in this case too, the last purification stage inevitably remains high-pressure liquid chromatography "HPLC" to eliminate extremely small impurities in the insulins. The disadvantages of reverse phase HPLC, such as risk of denaturation of the protein, bleeding of the RP silica gel phase, and unsatisfactory cleaning in place measures, must be accepted for this and require above-average expenditure of money and time.